Apoptosis, cell proliferation and expression of oncogenes in gastric carcinomas induced by preoperative administration of 5-fluorouracil.

The purpose of this study was to examine the correlations among enhancement of apoptosis, cell proliferation and expression of oncogenes in gastric carcinomas induced by preoperative oral administration of 5-fluorouracil (5-FU). The occurrence of spontaneous apoptotic cell death in 42 patients with gastric carcinoma was analyzed in the biopsy specimens preoperatively. p53 status was examined by polymerase chain reaction-single strand confirmation polymorphism and sequencing. Fourteen patients received oral administration of 5-FU at 300 mg/body/day for 7 days preoperatively. For detection of apoptotic cells, apoptotic incidences (AIs) were examined by the terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate biotin nick end labeling method, on gastric carcinoma lesions based on the endoscopic findings before administration in the biopsy and resected tissues. Expressions of p53, Bcl-2, Bax gene and proliferating cell nuclear antigen (PCNA) were also examined by immunohistochemical staining. On preoperative biopsy, p53 point mutation was observed in 14 of the 42 tumors. The immunohistochemical staining status and point mutation of p53 gene (positive or negative) were identical in 32 of the 42 tumors (76.2%). The average AIs of the biopsy specimens were 1.58+/-1.26% on p53-negative staining (n=19) and 1.14+/-1.02% on p53-positive staining (n=23), a significant association was not recognized between p53 expression and AI. In the preoperative administration group, the PCNA labeling index was significantly higher in the biopsy specimens than in the resected tissues (43. 6+/-12.8% vs. 35.3+/-8.8%, p<0.01). In addition, postoperatively, the rate of AI was significantly more accelerated in p53-negative staining (n=6) than in p53-positive staining (n=8) (0.89+/-0. 65%right curved arrow 4.18+/-3.26%, p<0.05 vs. 1.20+/-0.60%right curved arrow 2.60+/-2.60%, NS). There was no significant correlation between AI and Bcl-2 or Bax staining. Immunohistochemical analysis of p53 and PCNA stainings in biopsy specimens appears to be a well-characterized indicator of sensitivity of chemotherapy in gastric carcinomas.

Intratumoral genomic heterogeneity in human hepatocellular carcinoma detected by restriction landmark genomic scanning.

BACKGROUND/AIMS: One of the unique features of advanced hepatocellular carcinoma (HCC) is the morphological heterogeneity in a single tumor nodule. In order to investigate the intratumoral genomic heterogeneity of HCC, we performed Restriction Landmark Genomic Scanning (RLGS), which allows genomic DNAs to be surveyed at approximately 2000 landmark sites in a single 2-dimensional gel electrophoresis. METHODS: RLGS profiles of two regions from a single HCC nodule in six patients were compared with non-tumorous liver tissue. Four HCCs consisting of moderately-differentiated cells were separated into several small parts by thin fibrous septa, but not encapsulated. DNA samples were obtained from both parts of these so-called "nodule-in-nodule" HCC. Two HCCs consisting of well-differentiated cells which did not have a definite partition appeared pathologically homogeneous, and two independent regions of the HCC were used for the analysis. RESULTS: All six HCCs demonstrated different RLGS profiles (in total about 160 different spots) from the corresponding non-tumorous liver, and the number of different spots was greater in the 4 moderately-differentiated nodule-in-nodule HCCs (39-68 spots) than in the 2 well-differentiated homogeneous HCCs (6 and 3 spots). RLGS profiles of the two parts were different to each other in all 4 nodule-in-nodule HCCs. On the other hand, two other homogeneous HCCs showed the same RLGS profiles in the two regions. CONCLUSION: Thus, intratumoral genomic heterogeneity was demonstrated in the advanced HCC samples, and the genomic alterations may relate to the progression of HCC.

Clinical pilot study of intrahepatic arterial chemotherapy with methotrexate, 5-fluorouracil, cisplatin and subcutaneous interferon-alpha-2b for patients with locally advanced hepatocellular carcinoma.

To evaluate the efficacy of methotrexate (MTX)-5-fluorouracil (5-FU), cisplatin (CDDP), and interferon-alpha-2b(IFN alpha-2b) combination therapy, we conducted a clinical pilot study in patients with locally advanced hepatocellular carcinoma (HCC). Sixteen patients, who had received no prior treatment for the HCC, with portal tumor thrombosis in the main trunk or in the major branch were enrolled in the study. IFN alpha-2b (3 x 10(6) units) was injected subcutaneously 3 times per week. After a bolus administration of MTX (30 mg/m2), CDDP (75 mg/m2) and thereafter 5-FU (750 mg/m2) were given weekly by intrahepatic arterial infusion. In 15 eligible patients, there were 1 complete response (CR) and 6 partial responses (PR) with a response rate of 46.7%. Median survival of the 15 patients was 7 months, and the 2-year survival rate of CR and PR patients was 57.1%. There was severe transient hematologic toxicity. More than grade 2 nausea/vomiting was noted in > 50%. In conclusion, the IFN alpha-2b combination chemotherapy demonstrated good response in patients with locally advanced HCC. This treatment should be tried in a controlled study.

Involvement of peribiliary glands in primary sclerosing cholangitis: a histopathologic study.

We examined the histological changes of the peribiliary glands (PBGs), a hitherto pooly recognized anatomical element around the biliary tree, in 7 cases of primary sclerosing cholangitis (PSC). These glands showed proliferation, and nonspecific inflammation with lymphoplasmacytic infiltration, fibrosis, and destruction. In addition, there were cystic lesions around the bile ducts, and they were considered to reflect dilatation of the PBGs. These changes were found around the intrahepatic and extrahepatic bile ducts in the cases examined. It is of interest that changes in the PBGs tended to correlate with the inflammatory changes of the bile duct wall itself, though 2 cases showed changes in the duct walls and PBGs unrelated to their distribution along the biliary tree. These findings suggest that the PBGs are also a target structure in addition to the bile ducts themselves in PSC.

Transjugular intrahepatic portosystemic shunt after previous recanalization of a chronically thrombosed portal vein via a transmesenteric approach.

We report a cirrhotic patient with complete occlusion of the portal vein with marked cavernous transformation due to chronic thrombosis in whom a transjugular intrahepatic portosystemic shunt (TIPS) was successfully created after direct minilaparotomy mesenteric vein catheterization, lysis and aspiration of the thrombus, and stenting in the portal vein. The methods used, we believe, provide a new technique for performing TIPS in chronically thrombosed portal veins in which previously no effective surgical therapeutic options were available.

B-cell epitopes in hypervariable region 1 of hepatitis C virus obtained from patients with chronic persistent hepatitis.

The hypervariable domain (HVR1) within the N-terminus of the E2 protein of hepatitis C virus (HCV) is known to be variable antigenically during the course of persistent infection. The aim of the study was to detect B-cell epitopes in HVR1 responsible for neutralizing HCV. The B-cell epitopes were analyzed using two series of synthetic peptides: 25 heptapeptides from the most common amino acids within 73 HVR1 sequences, and 216 heptapeptides, the sequences of which cover more than 65% of the 73 HVR1 sequences. Sera from three patients with chronic hepatitis C were tested for reactivity to the synthetic peptide sequences by enzyme-linked immunosorbent assay (ELISA). The post-interferon (IFN) serum of one patient who had a long-term response to treatment reacted specifically with 13 heptapeptides of 216 variable sequences of HVR1. Some of the amino acid sequences (amino acids 398, 399, 400, 404) of the heptapeptides were also found in those deduced from the nucleotide sequences of HCV genomes in the pre-IFN serum. The sera of the other two patients who did not respond to treatment did not react with the 13 heptapeptides. It is concluded that the B-cell epitopes in HVR1 may be relevant for eliminating viremia in the case of the patient who had a good response to treatment. These results suggest that the analysis of the B-cell epitopes recognized in HVR1 may be important in understanding the mechanism of persistent infection and progression of hepatitis.

Prevention of endotoxin-induced acute lethality in Propionibacterium acnes-primed rabbits by an antibody to leukocyte integrin beta 2 with concomitant reduction of cytokine production.

Acute lethality was induced in rabbits by the sequential injection of Propionibacterium acnes and lipopolysaccharide (LPS). P. acnes induced the infiltration of inflammatory cells into the liver lobules during the early phase, and LPS in the late phase caused death in association with pathological changes mimicking hepatocellular necrosis or degeneration around infiltrated mononuclear cells and fibrin deposition in the liver, lung, and kidney, suggestive of a systemic Schwartzman-like reaction. These pathological changes were accompanied by the elevation of plasma tumor necrosis factor (TNF) and interleukin-8 (IL-8) levels. A neutralizing antibody to a leukocyte adhesion molecule, integrin beta 2 (CD18), administered at the time of LPS challenge, prevented reduced the elevation of plasma TNF and IL-8 levels. An anti-TNF alpha antibody but not an anti-IL-8 mediator in this model. These results indicate that CD18 is critically involved in vivo in activating leukocytes to produce cytokines in response to LPS.

Effects of chenodeoxycholic and ursodeoxycholic acids on interferon-gamma production by peripheral blood mononuclear cells from patients with primary biliary cirrhosis.

Ursodeoxycholic acid (UDCA) administration can obtain marked improvement of primary biliary cirrhosis (PBC). Recently, UDCA has been demonstrated to have a direct effect on immunological reactions in patients with PBC in that the aberrant expression of major histocompatibility complex (MHC) class I molecules was markedly reduced after UDCA treatment. To understand the immunological effect of UDCA, we analyzed interferon (IFN)-gamma production in peripheral blood mononuclear cells (PBMCs) from 29 patients with PBC treated with UDCA (group 1), 19 patients with PBC who were not treated with UDCA (group 2), 11 healthy subjects (group 3), and 12 patients with chronic viral hepatitis (group 4). IFN-gamma production was investigated because the excess production of this cytokine is associated with the aberrant expression of MHC molecules. Whereas IFN-gamma production in the patients in group 2 was significantly increased, the level of production in group 1 was similar to that in the control groups (groups 3 and 4). There was significant improvement in IFN-gamma production in 6 patients with PBC after UDCA treatment. The effect of UDCA and chenodeoxycholic acid (CDCA) on IFN-gamma production in PBMCs from 12 normal subjects was also analyzed. IFN-gamma was produced dose-dependently according to concentrations of CDCA ranging from 0.1 to 10 microM, but the increase in production was markedly suppressed by the addition of UDCA. We conclude that low doses of CDCA enhance IFN-gamma production and may therefore lead to the aberrant hepatic expression of MHC molecules, and that the increase in IFN-gamma production is suppressed by UDCA.

Hemosiderin deposition in portal endothelial cells: a novel hepatic hemosiderosis frequent in chronic viral hepatitis B and C.

We have recently noted a hitherto undescribed hepatic hemosiderosis confined to endothelial cells of the portal tract in chronic viral hepatitis. In this study, this lesion was surveyed in 156 liver biopsy specimens from patients with chronic hepatitis C and in 21 liver biopsy specimens from patients with chronic hepatitis B. As controls, we examined 110 liver biopsy specimens from patients with primary biliary cirrhosis (PBC), 36 from patients with alcoholic liver injury, nine from patients with autoimmune hepatitis (AIH), and five from patients with primary hemochromatosis. Hemosiderin deposition was found in the endothelial cells of venous vessels in portal tracts regardless of the presence or degree of hemosiderin deposition in hepatic parenchyma. This phenomenon was observed in 65 of 156 cases (42%) of chronic hepatitis C and in eight of 21 (38%) cases of chronic hepatitis B. In controls, this lesion was frequent in AIH (78%), but infrequent in PBC (8.1%) and alcoholic liver injury (11%). The incidence of this lesion showed significant differences between chronic hepatitis C, B, and AIH, and between PBC and alcoholic liver injury. There was a positive correlation between the progression of disease and the incidence of this feature in chronic viral hepatitis; the incidence was 18.3% and 11.1% in milder chronic hepatitis C and B, respectively, and 61.2% and 58.3%, respectively, in more severe cases. However, this correlation was not evident in either PBC or alcoholic liver injury. This hemosiderin deposition was positively correlated with the degree of piecemeal necrosis in chronic hepatitis C, and to a lesser degree, the positive correlation was shown in chronic hepatitis B. These findings suggest that the progression of chronic hepatitis and the piecemeal necrosis in chronic hepatitis C and B, followed by the release of hepatocellular iron to portal and periportal areas, are directly or indirectly responsible for endothelial hemosiderosis. Further studies focusing on this peculiar phenomenon in relation to choice of therapy and evaluation of chronicity of viral hepatitis are encouraged.

Three cases of primary biliary cirrhosis associated with bronchial asthma.

The association of primary biliary cirrhosis (PBC) and bronchial asthma was observed in three patients. All of these patients were female (53, 54, and 41 years old, respectively), and were positive for antimitochondrial antibodies. The patients fulfilled the diagnostic criteria of both PBC and bronchial asthma. Bronchial asthma preceded PBC in two patients, and the reverse order was seen in the other. Patient the clinical symptoms were mainly due to the bronchial asthma. Two patients had asymptomatic PBC and the third patient complained of pruritus. The liver histology showed mild to moderate eosinophilic infiltration in addition to the ductal and hepatic parenchymal changes characteristic of PBC. A survey of 266 cases of PBC referred to us disclosed that, in 6 of these, the PBC was associated with bronchial asthma, while no association with bronchial asthma was the material of found in 166 patients with viral hepatitis in our liver biopsy files. The 3 present cases we experienced suggest that bronchial asthma may be included in the list of extrahepatic diseases associated with PBC. The significance of this association is unclear and may merit further study. Steroid therapy, which is known to cause adverse effects in PBC, was employed for bronchial asthma in these 3 patients. Another therapeutic approach will have to be considered in patients with bronchial asthma associated with PBC.

Regulation of the alpha 2(I) collagen gene transcription in fat-storing cells derived from a cirrhotic liver.

Fat-storing cells (FSC) are the main producers of type I collagen in both normal and fibrotic livers. In order to elucidate the molecular mechanisms controlling collagen expression in FSC, we examined the transcription of the alpha 2(I) collagen gene (COL1A2) in two distinct FSC clones, CFSC-2G and CFSC-5H, derived from a single CCl4-induced cirrhotic liver. The phenotype of CFSC-2G resembles freshly isolated FSC, whereas that of CFSC-5H mimics activated myofibroblasts. Cell transfection experiments showed that the upstream sequence between nucleotides -378 and -183 is essential for COL1A2 transcription in both FSC clones. This is the same promoter region that is transcriptionally active and contains the binding site of a multimeric protein complex that mediates TGF-beta stimulation of COL1A2 expression in dermal fibroblasts. We therefore examined the relative levels of endogenous and transfected COL1A2 transcription and their response to TGF-beta treatment in the two FSC clones. The results showed that CFSC-5H expresses a significantly higher level of the COL1A2 mRNA than CFSC-2G. They also showed that TGF-beta treatment increases both endogenous and transfected COL1A2 transcription in CFSC-2G but not in CFSC-5H. Interestingly, nuclear proteins from both FSC clones bind to the TGF-beta-responsive element more strongly than those from dermal fibroblasts. Altogether, the data suggest that collagen production in CFSC-5H cells has been already activated by the autocrine stimulation of TGF-beta. In contrast, CFSC-2G cells are only partially activated but can be easily recruited to produce collagen when stimulated by exogenous TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon therapy in chronic hepatitis C virus: evidence of different outcome with respect to different viral strains.

The aim of the study was to assess the role of different viral strains of hepatitis C virus (HCV) in determining the outcome of the alpha-interferon (IFN) therapy. Fifty-seven patients (34 from Italy and 23 from Japan) with HCV-positive liver disease were enrolled in the study. The NS4 region of HCV was amplified in sera by "nested" polymerase chain reaction (PCR) using a primer pair synthesized according to the sequence of JK-1. The NS4 region was positive in 14 (41%) Italian and in 13 (56%) Japanese patients. In positive patients the sequence of the NS4 region was also obtained. Subsequently, HCV genotype was determined in all patients by PCR amplification of the core region. All patients received recombinant alpha 2a-interferon (IFN), 6 million units 3 times a week for 1 month followed by 3 million units 3 times a week for 5 months. The patients were followed for 1 year after the end of treatment. At the end of the follow-up, 17 (30%) had sustained normal levels of serum alanine aminotransferase (ALT). The outcome of treatment was not correlated with race, age, sex, histology, and pretreatment ALT level, but was significantly (P < 0.00001) associated with the presence of both the NS4-JK-1 region and HCV type II. Among the 27 NS4-positive patients, only 1 patient (3.7%) achieved a complete response, whereas the remaining 26 patients (95.3%) either were non-responders or relapsed after IFN was discontinued.(ABSTRACT TRUNCATED AT 250 WORDS)